Mechanisms of Toxicity

1. Mechanisms of Toxicity NST110, Toxicology Department of Nutritional Sciences and Toxicology University of California, Berkeley

2. Mechanisms of Toxicity • Delivery: Site of Exposure to the Target • Reaction of the Ultimate Toxicant with the Target Molecule • Cellular Dysfunction and Resultant Toxicity • Repair or Dysrepair

3. Mechanisms of Toxicity • Delivery: Site of Exposure to the Target • Reaction of the Ultimate Toxicant with the Target Molecule • Cellular Dysfunction and Resultant Toxicity • Repair or Dysrepair

4. Chemical Factors that Cause Cellular Dysfunction • Chemicals that cause DNA adducts can lead to DNA mutations which can activate cell death pathways; if mutations activate oncogenes or inactivate tumor suppressors, it can lead to uncontrolled cell proliferation and cancer (e.g. benzopyrene) • Chemicals that cause protein adducts can lead to protein dysfunction which can activate cell death pathways; protein adducts can also lead to autoimmunity; if protein adducts activate oncogenes or inactivate tumor suppressors, it can lead to uncontrolled cell proliferation and cancer (e.g. diclofenacglucuronidation metabolite) • Chemicals that cause oxidative stress can oxidize DNA or proteins leading to DNA mutations or protein dysfunction and all of the above. (e.g. benzene, CCl4) • Chemicals that specifically interact with protein targets • chemicals that activate or inactivate ion channels can cause widespread cellular dysfunction and cause cell death and many physiological symptoms—Na+, Ca2+, K+ levels are extremely important in neurotransmission, muscle contraction, and nearly every cellular function (e.g. tetrodotoxin closes voltage-gated Na+ channels) • Chemicals that inhibit cellular respiration—inhibitors of proteins or enzymes involved in oxygen consumption, fuel utilization, and ATP production will cause energy depletion and cell death (e.g. cyanide inhibits cytochrome c oxidase) • Chemicals that inhibit the production of cellular building blocks, e.g. nucleotides, lipids, amino acids (e.g. amanitin from Deathcap mushrooms) • Chemicals that inhibit enzymatic processes of bioactive metabolites that alter ion channels and metabolism (e.g. sarin inhibits acetylcholinesterase and elevates acetylcholine levels to active signaling pathways and ion channels) • All of the above can also cause inflammation which can lead to cellular dysfunction

5. Cellular Dysfunction: Necrosis versus Apoptosis

6. Two Forms of Cell Death • 1.Necrosis: unprogrammed cell death (dangerous) • A. Passive form of cell death induced by accidental damage of tissue and does not involve activation of any specific cellular program. • B. Early loss of plasma membrane integrity and swelling of the cell body followed by bursting of cell. • C. Mitochondria and various cellular processes contain substances that can be damaging to surrounding cells and are released upon bursting and cause inflammation. • D. Cells necrotize in response to tissue damage [injury by chemicals and viruses, infection, cancer, inflammation, ischemia (death due to blockage of blood to tissue)].

7. 2. Apoptosis: one of the main forms of programmed cell death (not as dangerous to organism as necrosis). • Active form of cell death enabling individual cells to commit suicide. • B. Caspase-dependent • C. Dying cells shrink and condense and then fragment, releasing small membrane-bound apoptotic bodies, which are phagocytosed by immune cells (i.e. macrophages). • D. Intracellular constituents are not released where they might have deleterious effects on neighboring cells.

9. Mechanisms of Apoptosis Apoptosis is a cell mechanism used to eliminate cells that contain mutations, are unnecessary, or dangerous to the body It is critical to normal embryonic development and to cancer prevention

10. Mechanisms of Apoptosis Phenotypes of apoptosis: Overall shrinkage in volume of the cell and its nucleus Loss of adhesion to neighboring cells Formation of blebs on the cell surface DNA fragmentation: dissection of the chromatin into small fragments Rapid engulfment of the dying cell by phagocytosis Factors that induce apoptosis: Internal stimuli: abnormalities in DNA External stimuli: removal of growth factors, addition of cytokines (tumor necrosis factor—TNF) Signal transduction pathways leading to apoptosis: Two major pathways: Intrinsic pathway (mitochondria-dependent) Extrinsic pathway (mitochondria-independent)

11. Extrinsic Apoptosis • The death receptor pathway I activated by external cytokines and is mitochondria-independent • The ligands of the death receptors are members of the tumor necrosis factor (TNF) family of proteins, including TNF-alpha, Fas ligand (FasL), TRAIL/Apo2L, Apo3L • Binding of ligand to the death receptors results in homotrimerization of the receptors • Death receptors contain a death domain in the cytoplasmic region that is required for apoptosis signaling

12. Extrinsic Apoptosis Trimerization of the receptor death domains allows binding and activation of FADD (Fas-associated death domain protein) and formation of death-inducing signaling complex (DISC), which recruits and activates procaspase 8 and 10 to caspase 8 and 10. Caspases are a family of cyteine-aspartyl-specific proteases that are activated at an early stage of apoptosis and are responsible for triggering most of the changes during apoptosis. Caspases are proteolytically activated and then diffuse into the cytoplasm to cleave target proteins

13. Extrinsic Apoptosis Two major classes of caspases: Initiator caspases 8,9,10: initiates the onset of apoptosis by activating the executioner caspases Initiator caspases 3,6,7: destroy actual targets in the cell to execute apoptosis Caspases target: FAK (focal adhesion kinase): inactivation of FAK disrupt cell adhesion, leading to detachment of the apoptotic cell from its neighbors Lamins: important component of the nuclear envelope, cleavage of lamins leads to disassembly of the nuclear lamina Proteins required for cell structure: actin, intermediate filaments, etc--cleavage of these proteins lead to changes in cell shape and the surface blebbing Endonuclease CAD: responsible for chromosome fragmentation. CAD cuts DNA into small fragments. CAD normally binds to an inhibitor protein. Caspases cleaves the inhibitor protein to activate CAD Enzymes involved in DNA repair

14. Extrinsic Apoptosis

15. Intrinsic Apoptosis Intrinsic apoptosis is mitochondria-dependent and is induced by DNA damage, binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, hypoxia, and increased intracellular calcium concentration Process of Intrinsic apoptosis: Bax forms homo-dimers in the presence of apoptotic signals, opening a channel that translocates cytochrome c from the intermembrane space to the cytoplasm Bcl2 interferes with Bax function by forming a heterodimer with Bax, closing the channel and inhibiting cytochrome c translocation In the cytosol, cytochrome c binds to Apaf-1 to form apoptosome Apoptosome recruits procaspase 9 and activates it to caspase 9 Caspase 9 activates executioner caspases 3, 6, and 7

16. Summary of Apoptosis

17. Mechanisms of Necrosis • Cells must synthesize endogenous molecules, assemble macromolecular complexes, membranes, and cell organelles, maintain intracellular environment, and produce energy for operation. • Agents that disrupt these functions (especially energy-producing function of the mitochondria and protein synthesis) will cause cell death.

18. ATP-SYN: ATP synthase MET: mitochondrial electron transport NOS: nitric oxide synthase PARP: poly(ADP-ribose) polymerase ROS: reactive oxygen species RNS: reactive nitrogen species XO: xanthine oxidase DYm: mitochondrial membrane potential Three Primary Metabolic Disorders Jeopardizing Cell Survival: I. ATP depletion II. Sustained rise in intracellular Ca2+ III. Overproduction of ROS, RNS

19. I. ATP Depletion • ATP plays a central role in cellular maintenance both as a chemical for biosynthesis and as the major source of energy. • ATP drives ion transporters such as Na+/K+-ATPase (plasma membrane), Ca2+ -ATPase (endoplasmic reticulum and plasma membrane) to maintain cellular ion gradients. (most important for necrosis!) • Used in biosynthetic reactions (phosphorylation and adenylation) • Used for signal transduction regulation (e.g. phosphorylation of receptor tyrosine kinase and kinase pathways) • Incorporated into DNA • Muscle contraction (actin/myosin interaction) and neurotransmission • Polymerization of cytoskeleton (actin and tubule polymerization) • Cell division • Maintenance of cell morphology

20. ATP Production in the Mitochondria

21. Direct Consequences of ATP Depletion

22. Agents That Impair ATP Synthesis • Inhibitors of electron transport • Cyanide inhibits cytochrome oxidase • Rotenone inhibits complex I—insecticide that may be an environmental cause of Parkinson’s Disease • Paraquat inhibits complex I—herbicide, but also causes lung hemorrhaging in humans • Inhibitors of oxygen delivery • Ischemic agents such as ergot alkaloids, cocaine • Carbon monoxide—displaces oxygen from hemoglobin • Inhibitors of ADP phosphorylation - DDT, DIM, phytochemicals • Chemicals causing mitochondrial DNA damage - antivirals, chronic ethanol

23. II. Sustained Rise of Intracellular Ca2+ Ca2+ is involved in : 1. signal transduction regulation (i.e. PKC activation by DAG and Ca2+) and exocytosis 2. muscle contraction (actin/myosin interaction) 3. cytoskeletal polymerization (i.e. Ca2+inhibition of actin) 4. neurotransmission (via glutamate receptor Ca2+channel and voltage-gated Ca2+channels) and synaptic plasticity 5. enzyme induction (i.e. citrate and -ketoglutarate dehydrogenases from the TCA cycle) 6. Transporters (Ca2+/ATPase, Na/Ca2+ exchanger, etc.)

24. Intracellular Ca2+ levels are highly regulated • The 10,000-fold difference between extracellular and cytosolic Ca2+ • concentration is maintained by: impermeability of plasma membrane • to Ca2+and by transport mechanisms that remove Ca2+from cytoplasm • (0.1 M inside versus 1000 M outside). • Ca2+ sources are from outside cell or Ca2+ stores in ER or • mitochondria (as calcium phosphate).

25. Four mechanisms of calcium elimination: 1. Extracellular Ca2+ ATPase 2. Endoplasmic reticulum Ca2+ ATPase 3. Extracellular Na+/Ca2+ exchanger 4. Mitochondrial Ca2+uniporter

27. Excitotoxicity: Consequence of Increased Intracellular Ca2+ • Depletion of energy reserves—decreased mitochondrial ATP production and increased loss of ATP by activation of Ca+2-ATPase. • Dysfunction of microfilaments—impaired cell motility, disruption in cell morphology, cellular functions • Activation of hydrolytic enzymes—disintegration of membranes, proteins, DNA, etc. • Generation of ROS/RNS—disintegration of membranes, proteins, DNA, etc.

28. III. Oxidative Stress Oxidative stress: imbalance of cellular oxidants and antioxidants in favor of oxidants.

29. Reactive Oxygen and Nitrogen Species Generation • A. Direct generation of ROS/RNS • a. Xenobiotic bioactivation (i.e. carbon tetrachloride, benzene) • b. Redox cycling (paraquat, MPP+) • c. Transition metals (Fe2+, Cu2+) • d. Inhibition of mitochondrial electron transport (many phytochemicals)

30. Reactive Oxygen Stress (ROS) and Reactive Nitrogen Species (RNS) Hydrogen peroxide peroxynitrite Superoxide Nitrosoperoxy carbonate Nitrogen dioxide Carbonate anion radical Hydroxyl radical

31. B. Indirect generation of ROS/RNS • a. Increased Ca2+ can cause ROS/RNS • Activates dehydrogenases in citric acid cycle and increases electron output (NADH and FADH2)leads to an increase in O2.- (superoxide) by the e- transport chain. • ii. Ca2+ -activated proteases proteolytically convert xanthine dehydrogenase to xanthine oxidase, the by-products of which are O2-. and H2O2. • iii. Neurons and endothelial cells constitutively express NOS that is activated by Ca2+increase .NO production which reacts with O2.-to produce highly reactive ONOO- (peroxynitrite). • b. Induction of CYPs (i.e. TCDD binding AhR)

32. Consequences of ROS/RNS • ROS can directly oxidize and affect protein function and can mutate DNA leading to cellular dysfunction • ROS/RNS oxidatively inactivate Ca2+ /ATPases and elevate Ca2+ • ROS and RNS also drain ATP reserves: • a. NO. is a reversible inhibitor of cytochrome oxidase • b. ONOO- irreversibly inactivates complexes I/II/III and aconitase • c. ROS can disrupt mitochondrial membranes and dissipate the electrochemical gradient needed for ATP synthase. • ONOO- induces DNA single-strand breaks, which activates poly(ADP-ribose) polymerase (PARP)—PARP transfers ADP-ribose moieties from NAD+ to PARP; consumption of NAD+ compromises ATP synthesis • Lipid peroxidation, cell swelling, and cell rupture

33. Lipid Peroxidation Free radicals can initiate peroxidative degradation of lipids by hydrogen abstraction from fatty acids. The lipid radical (L.) formed is converted to the lipid peroxyl radical (LOO.) by oxygen fixation lipid hydroperoxide (LOOH) is then formed by hydrogen abstraction from another lipid lipid alkoxyl radical (LO.) is formed by the Fe(II)-catalyzed Fenton reaction Fragmentation leads to reactive aldehydes, including the lipid aldehyde and free radicals Lipid peroxidation is auto-catalytic

35. Organophosphate (OP) Nerve Agents • Organophosphorus (OP) chemical warfare agents inhibit acetylcholinesterase (AChE) • Under the Nazi regime during World War II, OPs were developed as chemical warfare agents--they are also very easy to manufacture Before World War II, chemical warfare was revolutionized by Nazi Germany’s discovery of nerve agents tabun (in 1937) and sarin (in 1939) by Gerhard Schrader, a chemist of IG Farben. In 1952, researchers in Porton Down, England, invented the VX nerve agent.

36. Organophosphate (OP) Nerve Agents Act by Irreversibly Inhibiting Acetylcholinesterase OP

37. Normal Function of Acetylcholine and Acetylcholinesterase Acetylcholine binds to muscarinic ACh receptors on parasympathetic neurons—controls secretion (salivation, tearing, urination, digestion, defecation), heart rate, breathing Acetylcholine binds to nicotinic ACh receptors on cholinergic neurons—controls memory, motor function, neurotransmission

38. OP Poisoning OPs inhibit AChE, leading to accumulation of acetylcholine at the synapse. Excess acetylcholine hyperstimulates muscarinic ACh receptors leading to excess salivating, vomitting, tearing, urinating, defecating, bronchoconstriction, reduced heart rate, diarrhea Excess acetylcholine also hyperstimulates nicotinic ACh receptors leading to convulsionsand tremors

39. Example of Energy Depleting Neurotoxins: MPTP MPTP MPP+ • MPTP, a contaminant in desmethylprodine (MPPP), an opioid analgesic drug, gave several people in the 1970s and 1980s irreversible Parkinson’s Disease. • In 1976, Barry Kidston, a 23-year old graduate student in Maryland, synthesized MPPP with MPTP as a major contaminant and injected himself—developed full-blown Parkinson’s disease in 3 days—treated with levadopa but died 18 months later from cocaine overdose—autopsy revealed dopaminergic neurodegeneration

40. MPTP Causes Parkinson’s Disease Through Selective Degeneration of Dopaminergic Neurons in the SubstantiaNigra MPTP MPP+ • Mechanism of Action: • MPTP crosses the blood brain barrier • MPTP gets metabolized to the toxic bioactivated agent MPP+ by monoamine oxidase-B (MAOB) found in glial cells in the brain • MPP+ is selectively taken up by dopamine transporters in the brain • MPP+ inhibits complex I of the electron-transport chain and causes oxidative stress in dopaminergic neurons to cause neurodegeneration. • Over hours to days, patients develop irreversible symptoms of Parkinson’s disease, including tremor, hypokinesia, rigidity, and postural instability • Antidote: MAOB inhibitors such as selegiline are used as antidotes to prevent conversion of MPTP to MPP+

41. Repair Mechanisms DNA repair Protein repair Lipid repair

42. Oxidized Protein Repair • Protein disulfides (Prot-SS, Prot1-SS-Prot2), protein sulfenic acids (Prot-SOH) and protein methionine sulfoxides (Prot-Met=O) are reduced by thioredoxin (TR-[SH]2) or methionine sulfoxidereductase; thioredoxin is regenerated by thioredoxinreductase • Protein glutathione mixed disulfides (Prot-SSF) are reduced by glutaredoxin; glutaredoxin is regenerated by glutathione reductase

43. Peroxidized Lipid Repair • Phospholipid peroxyl radicals (PL-OO.) formed from lipid peroxidation may abstract hydrogens from alpha-tocopherol (TOC-OH), which can be regenerated by glutaredoxin (GRO), which in-turn can be regenerated by glutathione reductase (GR) • A phospholipase can cleave the fatty acid peroxide (FA-OOH), which can be reduced by glutathione peroxidase (GPX) to give FA-OH; GPX is regenerated by glutathione reductase

44. Quenching of Oxidative Stress Detoxification of superoxide anion radical occurs by superoxide dismutase (SOD), followed by glutathione peroxidase (GPO), and catalase (CAT).

45. DNA Repair Mechanisms

46. Inflammatory Response

47. Process of Acute Inflammation • Inflammatory pathway consists of inducers, sensors, mediators, and target tissues. • Inducers initiate the inflammatory response and are detected by sensors. • Sensors, like toll-like receptors (TLRs) are expressed on specialized sentinel cells such as macrophages, dendritic cells, and mast cells • TLRs recognize molecules broadly shared across pathogens (e.g. lipopolysaccharides, double-stranded RNA from viruses, bacterial flagella) • TLRs also recognize endogenous molecules associated with cell stress (e.g. fibrinogen involved in blood clotting), ATP, heat shock proteins (HSPs), HMBG1 involved in organizing DNA in the nucleosome, and self DNA

48. Process of Acute Inflammation • When activated, these sensing cells secrete inflammatory mediators including cytokines (e.g. tumor necrosis factor-alpha (TNFa), interleukin-1-beta (IL-1b), and IL-6), chemokines (e.g. CCL2, CXCL8), bioactive amines (e.g. histamine), bradykinin, inflammatory lipids (eicosanoids) • These inflammatory mediators dilate blood vessels, recruit more immune cells, and act on target tissues to eliminate the inflammatory agent, repair the tissue, and elicit changes in their functional states that optimizes their response to noxious conditions

49. TNF Signaling and Effects TNF binds to TNF receptors, causing the receptor to form a trimer that recruits TRADD, and can activate 3 pathways: Activation of NF-kB: TRADD recruits TRAF2 and RIP, TRAF2 recruits protein kinase IKK, which is then activated by RIP. IKK phosphorylates IkBa, which releases NFkB to translocate to the nucleus to act as a transcriptional activator of genes involved in cell survival, proliferation, inflammation, and anti-apoptotic factors Activation of MAPK pathways: TNF induces activation of p38-MAP kinase signaling through activation of ASK1 and MEKK1, eventually leading to the phosphorylation of MKK7 which activates JNK, which is translocated to the nucleus and activates the AP-1 transcription factor to induce cell differentiation and proliferation genes Induction of death signaling: TNF can also induce cell death through TRADD recruiting FAS-associated protein with death domain (FADD), which recruits caspase 8, a protease that activates caspase 3, leading to apoptosis Cell death Cell proliferation survival Inflammation, Proliferation, survival Whether a cell undergoes proliferation/inflammation or cell death depends on overall inflammatory environment (other cytokines or ROS).

50. TNF Signaling and Effects TNF stimulation leads to: Fever Chemoattractant for neutrophils Stimulates macrophage activation and phagocytosis Production of oxidative stress Production of other inflammatory mediators like eicosanoids Causes insulin resistance Cell death Cell proliferation survival Inflammation, Proliferation, survival